Reversible inhibitors of SAH hydrolase and uses thereof

ABSTRACT

The present invention provides compositions and methods for reversibly inhibiting S-adenosyl-L-homocysteine (SAH) hydrolase. The compounds of the present invention can be used in combination with an anti-hemorrhagic viral infection agent, an immunosuppressant, a homocysteine lowering agent, or an anti-neoplasm agent. The compositions and methods of the present invention can be used for the prevention and treatment of hemorrhagic virus infection, autoimmune diseases, autograft rejection, neoplasm, hyperhomocysteineuria, cardiovascular disease, stroke, Alzheimer&#39;s disease, or diabetes.

BACKGROUND OF THE INVENTION

[0001] SAH hydrolase has been an attractive target for antiviral drugdesign based on the observation that many viruses require 5′-capped,methylated structures on their mRNA for efficient translation of viralproteins. Yuan et al., Exp. Opin. Ther. Patents, 9: 1197-1206 (1999);Yuan et al., in Adv. Antiviral Drug Des. vol 2, pp. 41-88, De Clercq(ed)., JAI Press, Inc. London, UK (1996). Inhibition of SAH hydrolaseresults in inhibition of S-adenosyl-L-methionine (SAM)-dependentmethylation reactions, including viral mRNA methylation, thus inhibitingviral replication (Scheme 1).

[0002] Scheme 1. Mechanism of methylation based inhibition of viralreplication

[0003] Numerous inhibitors of SAH hydrolase have been identified fromnaturally occurring compounds and synthetic compounds. Most potentinhibitors are irreversible inhibitors, which irreversibly inactivateSAH hydrolase in a time-dependent fashion. Studies have demonstratedthat irreversible inhibitors only produce narrow therapeutic windows dueto their severe cytotoxic effects (Wolfe and Borchardt, Journal ofMedicinal Chemistry, 34:1521-1530 (1991)). Since SAH hydrolase is aubiquitous cellular enzyme with a very slow turnover rate (t_(1/2) =24hours in mouse liver), irreversible inhibitors can cause prolongedinhibition of the enzyme activity. For instance, it can take up to sevendays for complete recovery of enzyme activity, which can lead tounwanted side effects. The severe cytotoxicity associated withirreversible inhibitors has been the major factor that has impaired thedevelopment of these inhibitors into clinically useful drugs. Because ofthe cytotoxicity associated with irreversible inhibitors, reversibleinhibitors are preferred.

[0004] However, at present, there are no known reversible SAH hydrolaseinhibitors that are potent enough to produce substantial inhibitoryactivity against the enzyme when tested in vivo. For example, thereversible inhibitor (S)-9-(2,3-dihydroxypropyl)adenine ((S)-DHPA),which has a K_(i) value of 3.5 μM against SAH hydrolase, lacksinhibitory potency. (Votruba and Holy, Coll. Czech. Chem. Commun.,45:3039 (1980)). Though (s)-DHPA was reported to be a reversibleinhibitor of isolated AdoHcy hydrolase (Votruba and Holy, Coll. Czech.Chem. Commun., 45:3039 (1980)), it was also reported to be airreversible inhibitor of intracellular AdoHcy hydrolase (Schanche etal., Molecular Pharmacology, 26:553-558 (1984)). Thus, existingreversible inhibitors are not clinically useful therapeutic agents, andthere remains a need for SAH hydrolase inhibitors that exhibit potencywithout the undesired cytotoxic effects.

BRIEF SUMMARY OF THE INVENTION

[0005] The present invention provides novel reversible inhibitors of SAHhydrolase. The compounds of the present invention are useful as agentsdemonstrating biological activities related to their ability to inhibitSAH hydrolase. In one embodiment, the reversible inhibitors of SAHhydrolase have the formula (I), and pharmaceutically acceptable saltsthereof:

[0006] wherein Z is carbon or nitrogen, R1 and R2 are the same ordifferent, and are hydrogen, hydroxy, alkyl, cycloalkyl, alkenyl,alkoxy, amino, aryl, heteroaryl, or halogen; R3 and R4 are the same ordifferent and are hydrogen, alkyl, acetyl, alkenyl, aryl, or heteroaryl;X is oxygen, nitrogen, or sulfur; and Y is hydrogen, a C₁₋₁₀ alkylgroup, alkenyl, vinyl, aryl, or heteroaryl. In a particular embodiment,the compound is not (4-adenine-9-yl)-2-hydroxybutanoic acid.

[0007] Compounds of formula I can have an S configuration at the βcarbon, an R configuration at the β carbon, or comprise a racemicmixture. In one embodiment, the compounds have a K_(i) value less than100 nM for a mammalian SAH hydrolase in a biological medium, e.g.,serum. In other embodiments, the compounds have a K_(i) value betweenabout 1 nM and about 100 nM for a mammalian SAH hydrolase in abiological medium. The compounds preferably have a K_(i) value less than100 nM, or a K_(i) value between about 1 nM and about 100 nM for a humanSAH hydrolase in a biological medium.

[0008] Compounds of formula I can have substituents wherein R1, R2, R3,and R4 are hydrogen. In one aspect of the present invention, X isoxygen. In another aspect of the present invention, Y is hydrogen or aC₁₋₁₀ alkyl group. In yet another aspect of the present invention, R1,R2, R3, and R4 are hydrogen, X is oxygen, and Y is hydrogen or a C₁₋₁₀alkyl group.

[0009] The present invention also relates to a pharmaceuticalcomposition comprising an effective amount of a compound of formula I orpharmaceutically acceptable salts thereof, and a pharmaceuticallyacceptable carrier or diluent. Pharmaceutical compositions may beadministered by oral, parenteral (e.g., intramuscular, intraperitoneal,intravenous, intracistemal injection or infusion, subcutaneousinjection, or implant), inhalation spray, nasal, vaginal, rectal,sublingual, or topical routes of administration. The pharmaceuticalcompositions may be formulated in suitable dosage unit formulationsappropriate for each route of administration.

[0010] It is not intended that the present invention be limited toparticular formulations or particular modes of administration. In oneembodiment, the composition is formulated for oral, parenteral,intranasal, topical, or injectable administration. Non-limited examplesof injectable administration are intracavemous injection, subcutaneousinjection, intravenous injection, intramuscular injection andintradermal injection. The pharmaceutical composition can be formulatedfor oral administration in a dosage ranging from about 0.1 to about 20mg/kg per day. The pharmaceutical composition can also be formulated forinjectable administration in a dosage ranging from about 0.1 to about 20mg/kg per day.

[0011] Pharmaceutical compositions of the present invention can beformulated in a solid or liquid dosage form. For example, thepharmaceutical compositions may be formulated as a solid in the form oftablets, capsules, granules, powders, and similar compounds. Thepharmaceutical compositions may also be formulated as a liquid in theform of syrups, injection mixtures, and the like.

[0012] The present invention also provides a kit comprising an effectiveamount of the composition of the present invention, and an instructionmeans for administering the composition.

[0013] Furthermore, the present invention provides methods forreversibly inhibiting the activity of a S-adenyl-L-homocysteine (SAH)hydrolase. In one embodiment, the present invention provides a methodfor reversibly inhibiting activity of a S-adenosyl-L-homocysteine (SAH)hydrolase in a mammal, comprising administering to a mammal to whichsuch reversible inhibition is needed or desirable, an effective amountof a compound or a pharmaceutically acceptable salt thereof, having theformula (I):

[0014] wherein Z is selected from the group consisting of carbon andnitrogen, R1 and R2 are the same or different, and are selected from thegroup consisting of hydrogen, hydroxy, alkyl, cycloalkyl, alkenyl,alkoxy, amino, aryl, heteroaryl, and halogen; R3 and R4 are the same ordifferent and are selected from the group consisting of hydrogen, alkyl,acetyl, alkenyl, aryl, and heteroaryl; X is selected from the groupconsisting of oxygen, nitrogen, and sulfur; and Y is selected from thegroup consisting of hydrogen, a C₁₋₁₀ alkyl group, alkenyl, vinyl, aryl,and heteroaryl, thereby reversibly inhibiting the activity of SAHhydrolase in said mammal. In a particular embodiment, the administeredcompound or a pharmaceutically acceptable derivative thereof is not(4-adenine-9-yl)-2-hydroxybutanoic acid.

[0015] In preferred embodiments, the mammal is suspected of having adisease selected from the group consisting of hemorrhagic viralinfection, autoimmune disease, autograft rejection, neoplasm,hyperhomocysteineuria, cardiovascular disease, stroke, Alzheimer'sdisease, diabetes, inflammatory Bowel disease, multiple sclerosis andautoimmune neuritis. However, it is not intended that the presentinvention be limited to the prevention and treatment of particulardiseases.

[0016] It is an object of the present invention to provide methods forpreventing and treating hemorrhagic viral infections. In one aspect, themethod comprises administering an effective amount of compounds havingformula I in the treatment of hemorrhagic viral infections in a mammal.In particular embodiments, the hemorrhagic viral infection is caused bya virus selected from the group consisting of a Bunyaviridaea, aFiloviridae, a Flaviviridae, and an Arenaviridae virus. In otherparticular embodiments, the Filoviridae virus is Ebola virus.

[0017] It is also an object of the present invention to provide methodsfor preventing and treating autoimmune diseases. In one aspect, themethod comprises administering an effective amount of compounds havingformula I in the treatment of an autoimmune disease in a mammal.

[0018] It is also an object of the present invention to provide methodsfor preventing and treating allograft rejection. In one aspect, themethod comprises administering an effective amount of compounds havingformula I in the treatment of allograft rejection in a mammal.

[0019] Furthermore, it is an object of the present invention to providemethods for preventing or treating hyperhomocysteineuria, or forlowering plasma homocysteine in a mammal. In one aspect, the methodcomprises administering an effective amount of compounds having formulaI for lowering plasma homocysteine in a mammal.

[0020] Further, it is an object of the present invention to providemethods for preventing or treating neoplasm. In one aspect, the methodcomprises administering an effective amount of compounds having formulaI for in the treatment of neoplasm in a mammal. Non-limiting examples ofneoplasm are neoplasm of the adrenal gland, anus, auditory nerve, bileducts, bladder, bone, brain, breast, bruccal, central nervous system,cervix, colon, ear, endometrium, esophagus, eye, eyelids, fallopiantube, gastrointestinal tract, head and neck, heart, kidney, larynx,liver, lung, mandible, mandibular condyle, maxilla, mouth, nasopharynx,nose, oral cavity, ovary, pancreas, parotid gland, penis, pinna,pituitary, prostate gland, rectum, retina, salivary glands, skin, smallintestine, spinal cord, stomach, testes, thyroid, tonsil, urethra,uterus, vagina, vestibulocochlear nerve, and the vulva.

[0021] The present invention also provides a combination, comprising aneffective amount of a compound having formula I, and an effective amountof an anti-hemorrhagic viral infection agent, an immunosuppressant, aplasma homocysteine lowering agent, and an anti-neoplasm agent. Thecombination can further comprise a pharmaceutically acceptable carrieror excipient. In a particular embodiment, the combination does notinclude (4-adenine-9-yl)-2-hydroxybutanoic acid.

[0022] In a particular embodiment, the anti-hemorrhagic viral infectionagent inhibits interleukin-1 (IL-1), tumor necrosis factor (TNF), or acombination thereof. The anti-hemorrhagic viral infection agent can bean anti-viral vaccine, an anti-viral antibody, a viral-activated immunecell, or a viral-activated immune serum.

[0023] In another embodiment, the immunosuppressant is cyclosporine,tacrolimus, an adrenocortical steroid, azathioprine, mycophenolate,cyclophosphamide, methotrexate, chlorambucil, vincristine, vinblastine,dactinomycin, an antithymocyte globulin, muromonab-CD3 monoclonalantibody, Rh₀(D) immunoglobulin, methoxsalen, or thalidomide.

[0024] In other particular embodiments, the homocysteine lowering agentis vitamin B₆, vitamin B₁₂, or folate.

[0025] In yet other embodiments, the anti-neoplasm agent is ananti-angiogenic agent, an alkylating agent, an antimetabolite, a naturalproduct, a platinum coordination complex, an anthracenedione, asubstituted urea, a methylhydrazine derivative, an adrenocorticalsuppressant, a hormone, an antagonist, an oncogene inhibitor, a tumorsuppressor gene or protein, an anti-oncogene antibody, or ananti-oncogene antisense oligonucleotide.

[0026] The present invention also provides a kit comprising an effectiveamount of the combination of the present invention, and an instructionmeans for administering the combination.

[0027] Furthermore, the present invention provides a method forreversibly inhibiting activity of a SAH hydrolase in a mammal,comprising administering to a mammal to which such reversible inhibitionis needed or desirable, an effective amount of a combination, whereinthe combination comprises: a) an effective amount of a compound or apharmaceutically acceptable salt thereof, having the formula (I):

[0028] wherein Z is selected from the group consisting of carbon andnitrogen, R1 and R2 are the same or different, and are selected from thegroup consisting of hydrogen, hydroxy, alkyl, cycloalkyl, alkenyl,alkoxy, amino, aryl, heteroaryl, and halogen; R3 and R4 are the same ordifferent and are selected from the group consisting of hydrogen, alkyl,acetyl, alkenyl, aryl, and heteroaryl; X is selected from the groupconsisting of oxygen, nitrogen, and sulfur; and Y is selected from thegroup consisting of hydrogen, a C₁₋₁₀ alkyl group, alkenyl, vinyl, aryl,and heteroaryl; and b) an effective amount of a compound selected fromthe group consisting of an anti-hemorrhagic viral infection agent, animmunosuppressant, a homocysteine lowering agent, and an anti-neoplasmagent, thereby reversibly inhibiting said activity of SAH hydrolase insaid mammal. In a particular embodiment, the administered combinationdoes not include (4-adenine-9-yl)-2-hydroxybutanoic acid.

[0029] The combination can be used with any other pharmaceuticalcomposition to modulate SAH hydrolase activity in a mammal. Thecombination can also be used in the prevention and treatment of diseasessuch as hemorrhagic viral infection, autoimmune disease, autograftrejection, neoplasm, and hyperhomocysteineuria, cardiovascular disease,stroke, Alzheimer's disease, diabetes, inflammatory Bowel disease,multiple sclerosis or autoimmune neuritis, as described above. However,it is not intended that the combination be limited to the prevention anduses of particular diseases.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0030]FIG. 1 illustrates effects of DZ2002 on Quantitative hemolysis ofSheep Red Blood Cells (QHS) assay. Data were expressed as means±SD.**:P<0.01 compared with control.

[0031]FIG. 2 illustrates that DZ2002 suppresses T cell proliferation inmixed lymphocyte reaction. Data were expressed as means±SD.***: P<0.001compared with control.

[0032]FIG. 3 illustrates that DZ2002 have no cytotoxicity in spleencell.

[0033]FIG. 4 illustrates effects of DZ2002 on DTH ear swelling in Balb/cmice.

[0034]FIG. 5 illustrates effects of DZ2002 on TNF-α production from TGinduced peritoneal cells.

[0035]FIG. 6A illustrates effects of DZ2002 on the expression of MHC-IIon THP-1 cells; FIG. 6B illustrates effects of DZ2002 on the expressionof CD80 on THP-1 cells; and FIG. 6C illustrates effects of DZ2002 on theexpression of CD86 on THP-1 cells.

[0036]FIGS. 7A and 7B illustrate effects of DZ2002 on IL-12P40 andIL-112P70 production from THP-1 cells.

DETAILED DESCRIPTION OF THE INVENTION

[0037] For clarity of disclosure, and not by way of limitation, thedetailed description of the invention is divided into the subsectionsthat follow.

[0038] A. Definitions

[0039] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as is commonly understood by one ofordinary skill in the art to which this invention belongs. All patents,applications, published applications and other publications referred toherein are incorporated by reference in their entirety. If a definitionset forth in this section is contrary to or otherwise inconsistent witha definition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth in this section prevails over thedefinition that is incorporated herein by reference.

[0040] As used herein, “a” or “an” means “at least one” or “one ormore.”

[0041] As used herein, a “composition” refers to any mixture of two ormore products or compounds. It may be a solution, a suspension, liquid,powder, a paste, aqueous, non-aqueous, or any combination thereof.

[0042] As used herein, a “combination” refers to any association betweentwo or among more items.

[0043] As used herein, “homocysteine” (Hcy) refers to a compound withthe following molecular formula: HSCH₂CH₂CH(NH₂)COOH. Biologically, Hcyis produced by demethylation of methionine and is an intermediate in thebiosynthesis of cysteine from methionine. The term “Hcy” encompassesfree Hcy (in the reduced form) and conjugated Hcy (in the oxidizedform). Hcy can conjugate with proteins, peptides, itself or other thiolsthrough a disulfide bond.

[0044] As used herein, “SAH hydrolase” refers to an enzyme whichcatalyzes hydrolysis of SAH to adenosine (Ado) and Hcy. The enzyme is anubiquitous eukaryotic enzyme, which is also found in some prokaryotes.SAH hydrolase also catalyzes the formation of SAH from Ado and Hcy. Theco-enzyme of SAH hydrolase is NAD⁺/NADH. SAH hydrolase may have severalcatalytic activities. In the hydrolytic direction, the first stepinvolves oxidation of the 3′-hydroxyl group of SAH (3′-oxidativeactivity) by enzyme-bound NAD⁺(E-NAD⁺), followed by β-elimination ofL-Hcy to give 3′-keto-4′,5′-didehydro-5′-deoxy-Ado. Michael addition ofwater to the 5′-position to this tightly bound intermediate(5′-hydrolytic activity) affords 3′-keto-Ado, which is then reduced byenzyme-bound NADH (E-NADH) to Ado (3′-reduction activity). It isintended to encompass SAH hydrolase with conservative amino acidsubstitutions that do not substantially alter its activity.

[0045] As used herein, the terms “pharmaceutically acceptable salts” or“pharmaceutically acceptable derivatives” of the compounds of thepresent invention encompass any salts, esters or derivatives that may bereadily prepared by those of skill in this art. Pharmaceuticallyacceptable salts of the compounds of this invention include, forexample, those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Salts derived from appropriate bases include,but are not limited to, alkali metal (e.g., sodium), alkaline earthmetal (e.g., magnesium), ammonium and N(C₁₋₄ alkyl)₄ ⁺ salts. Examplesof suitable acids include, but are not limited to, hydrochloric,hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric,glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric,acetic, citric, methanesulfonic, formic, benzoic, malonic,naphthalene-2-sulfonic, and benzenesulfonic acids. Other acids, such asoxalic, while not in themselves pharmaceutically acceptable, may beemployed in the preparation of salts useful as intermediates inobtaining the compounds of the invention and their pharmaceuticallyacceptable acid salts.

[0046] As used herein, “biological activity” refers to the in vivoactivities of a compound or physiological responses that result upon invivo administration of a compound, composition, or other mixture.Biological activity, thus, encompasses therapeutic effects andpharmaceutical activity of such compounds, compositions and mixtures.Biological activities may be observed in in vitro systems designed totest or use such activities.

[0047] As used herein, “plasma” refers to the fluid, noncellular portionof the blood, distinguished from the serum obtained after coagulation.

[0048] As used herein, “serum” refers to the fluid portion of the bloodobtained after removal of the fibrin clot and blood cells, distinguishedfrom the plasma in circulating blood.

[0049] As used herein, “fluid” refers to any composition that can flow.Fluids thus encompass compositions that are in the form of semi-solids,pastes, solutions, aqueous mixtures, gels, lotions, creams, and othersuch compositions.

[0050] As used herein, the abbreviations for any protective groups,amino acids and other compounds, are in accord with their common usage,recognized abbreviations, or the IUPAC-IUB Commission on BiochemicalNomenclature, unless otherwise indicated (see Biochemistry 11: 1726(1972)).

[0051] As used herein, “disease or disorder” refers to a pathologicalcondition in an organism, which is characterizable by identifiablesymptoms.

[0052] As used herein, the term “a therapeutic agent” refers to anyconventional drug or drug therapies which are known to those skilled inthe art, including, but not limited to vaccines.

[0053] As used herein, “vaccine” refers to any compositions intended foractive immunological prophylaxis. A vaccine may be used therapeuticallyto treat a disease, to prevent development of a disease, or to decreasethe severity of a disease either proactively or after infection.Exemplary vaccines include, but are not limited to, preparations ofkilled microbes of virulent strains, living microbes of attenuated(variant or mutant) strains, or microbial, fungal, plant, protozoa, ormetazoa derivatives or products. The term also encompassesprotein/peptide and nucleotide based vaccines.

[0054] As used herein, the term “therapeutically effective amount”refers to that amount that is sufficient to ameliorate, or in somemanner reduce the symptoms associated with the disease. Such amount maybe administered as a single dosage or according to a regimen. Repeatedadministration may be required to achieve the desired amelioration ofsymptoms.

[0055] As used herein, the terms “administration” or “administering” acompound refers to any suitable method of providing a compound of theinvention or a pro-drug of a compound of the invention to a subject.

[0056] As used herein, the term “treatment” refers to any manner inwhich the symptoms of a condition, disorder or disease are amelioratedor otherwise beneficially altered. Treatment also encompasses anypharmaceutical use of the compositions herein. Amelioration of symptomsof a particular disorder refers to any lessening of symptoms, whetherpermanent or temporary, that can be attributed to or associated withadministration of the composition.

[0057] As used herein, the term “substitute” refers to the replacementof a hydrogen atom in a compound with a substituent group.

[0058] As used herein, the term “alkyl” encompasses straight or branchedalkyl groups, including alkyl groups that are optionally substitutedwith one or more substituents. For example, the alkyl group can beoptionally substituted with hydroxy, halogen, aryl, alkoxy, acyl, orother substituents known in the art. One of more carbon atoms of thealkyl group can also be optionally replaced by one or more heteroatoms.

[0059] As used herein, the term “K_(i)” refers to a numerical measure ofthe effectiveness of a compound in inhibiting the activity of a targetenzyme such as ICE. Lower values to K_(i) reflect higher effectiveness.The K_(i) value is derived by fitting experimentally determined ratedata to standard enzyme kinetic equations (Segel, Enzyme Kinetics,Wiley-Interscience, 1975).

[0060] As used herein, “an anti-neoplastic treatment” refers to anytreatment designed to treat the neoplasm, tumor or cancer by lesseningor ameliorating its symptoms. Treatments that prevent the occurrence orlessen the severity of neoplasm, tumor or cancer are also contemplated.

[0061] As used herein, “neoplasm (neoplasia)” refers to abnormal newgrowth, and thus means the same as tumor, which may be benign ormalignant. Unlike hyperplasia, neoplastic proliferation persists even inthe absence of the original stimulus.

[0062] As used herein, “an anti-neoplasm agent (used interchangeablywith anti-neoplastic agent, anti-tumor or anti-cancer agent)” refers toany agents used in the anti-neoplasm treatment. These include anyagents, that when used alone or in combination with other compounds, canalleviate, reduce, ameliorate, prevent, place or maintain in a state ofremission clinical symptoms or diagnostic markers associated withneoplasm, tumor or cancer. The anti-neoplasm agent that can be used inthe combinations of the present invention include, but are not limitedto, anti-angiogenic agents, alkylating agents, antimetabolite, certainnatural products, platinum coordination complexes, anthracenediones,substituted ureas, methylhydrazine derivatives, adrenocorticalsuppressants, certain hormones and antagonists, anti-cancerpolysaccharides, and certain herb extracts such as Chinese herbextracts.

[0063] As used herein, “tumor suppressor gene” (also referred to asanti-oncogene or cancer susceptibility gene) refers to a gene thatencodes a product which normally negatively regulates the cell cycle,and which must be mutated or otherwise inactivated before a cell canproceed to rapid division. Exemplary tumor suppressor genes include, butare not limited to, p16, p21, p53, RB (retinoblastoma), WT-1 (Wilm'stumor), DCC (deleted in colonic carcinoma), NF-1 (neurofibrosarcoma) andAPC (adenomatous polypospis coli).

[0064] As used herein, “oncogene” refers to a mutated and/oroverexpressed version of a normal gene of animal cells (theproto-oncogene) that in a dominant fashion can release the cell fromnormal restraints on growth. Thus, an oncogene alone, or in concert withother changes, converts a cell into a tumor cell. Exemplary oncogenesinclude, but are not limited to, abl, erbA, erbB, ets, fes (fps), fgr,fms, fos, hst, int1, int2, jun, hit, B-lym, mas, met, mil (raj), mos,myb, myc, N-myc, neu (ErbB2), ral (mil), Ha-ras, Ki-ras, N-ras, rel,ros, sis, src, ski, trk and yes.

[0065] As used herein, “antisense polynucleotides” refer to syntheticsequences of nucleotide bases complementary to mRNA or the sense strandof double stranded DNA. Admixture of sense and antisense polynucleotidesunder appropriate conditions leads to the binding of the two molecules,or hybridization. When these polynucleotides bind to (hybridize with)mRNA, inhibition of protein synthesis (translation) occurs. When thesepolynucleotides bind to double stranded DNA, inhibition of RNA synthesis(transcription) occurs. The resulting inhibition of translation and/ortranscription leads to an inhibition of the synthesis of the proteinencoded by the sense strand.

[0066] As used herein, “antibody” includes antibody fragments, such asFab fragments, which are composed of a light chain and the variableregion of a heavy chain.

[0067] As used herein, “humanized antibodies” refers to antibodies thatare modified to include “human” sequences of amino acids so thatadministration to a human will not provoke an immune response. Methodsfor preparing such antibodies are known. For example, the hybridoma thatexpresses the monoclonal antibody is altered by recombinant DNAtechniques to express an antibody in which the amino acid composition ofthe non-variable regions is based on human antibodies. Computer programshave been designed to identify such regions.

[0068] As used herein, “anti-hemorrhagic virus agent” or “anti-viralhemorrhagic agent” refer to any agent used in the treatment ofhemorrhagic viral infections. These include any agents, alone or incombination with other compounds, that can alleviate, reduce,ameliorate, prevent, or maintain in a place of remission clinicalsymptoms or diagnostic markers associated with viral hemorrhagicdiseases, or disorders. Non-limiting examples of antiviral-hemorrhagicagents include interleukin-1 (IL-1) inhibitors, tumor necrosis factor(TNF) inhibitors, anti-viral vaccines, anti-viral antibodies,viral-activated immune cells, and viral-activated immune sera.

[0069] As used herein, “an anti-hemorrhagic virus treatment” refers toany treatment designed to treat hemorrhagic viral infections bylessening or ameliorating the symptoms. Treatments that prevent theinfection or lessen its severity are also contemplated.

[0070] As used herein, “IL-1 inhibitor” encompasses any substances thatprevent or decrease production, post-translational modifications,maturation, or release of IL-1, or any substances that interfere with ordecrease the efficacy of the interaction between IL-1 and IL-1 receptor.Preferably, the IL-1 inhibitor is an anti-IL-1 antibody, an anti-IL-1receptor antibody, an IL-1 receptor antagonist, an IL-1 productioninhibitor, an IL-1 receptor production inhibitor, or an IL-1 releasinginhibitor.

[0071] As used herein, “tumor necrosis factor” (“TNF”) refers to a groupof proinflammatory cytokines encoded within the major histocompatibilitycomplex. The TNF family members include TNF α and TNFR (also known ascachectin and lymphotoxin, respectively). Complementary cDNA clonesencoding TNA α and TNFR have been isolated. Thus, reference to “TNF”encompasses all proteins encoded by the TNF gene family, including TNF αand TNF, or an equivalent molecule obtained from any other source orthat has been prepared synthetically. It is intended to encompass TNFwith conservative amino acid substitutions that do not substantiallyalter its activity.

[0072] As used herein, “TNF inhibitor” encompasses any substances thatprevent or decrease production, post-translational modifications,maturation, or release of TNF, or any substances that interfere with ordecrease the efficacy of the interaction between TNF and TNF receptor.Preferably, the TNF inhibitor is an anti-TNF antibody, an anti-TNFreceptor antibody, a TNF receptor antagonist, a TNF productioninhibitor, a TNF receptor production inhibitor, or a TNF releasinginhibitor.

[0073] B. Reversible Inhibitors of S-Adenosyl-L-Homocysteine Hydrolase

[0074] One approach for minimizing mechanism-based cytotoxicity is tooptimize the pharmacokinetic profiles of SAH hydrolase inhibitors, suchthat the inhibitors exhibit reversible inhibiting activity.Pharmacokinetic profiles can be optimized by optimizing K_(off) values.For example, K_(off) values are optimized such that they are smallenough to produce desired antiviral effects, but large enough to allowadequate recovery of the enzyme activity before the next dose.

[0075] Eritadenine Derivatives as Reversible Inhibitors of SAH Hydrolase

[0076] The present invention relates to novel inhibitors ofS-adenosyl-L-homocysteine compositions that are reversible and potent.For example, the present invention provides compounds with a K_(i) valueof less than 100 nM. In one embodiment, the present invention provides4(adenine-9-yl)-2-hydroxybutanoic acid, its derivatives, andpharmaceutically acceptable salts thereof, and methods for reversiblyinhibiting SAH hydrolase using such compounds.

[0077] The reversible inhibitor, 4(adenine-9-yl)-2-hydroxybutanoic acidis synthesized from deoxyl modification of eritadenine at the betacarbon. Eritadenine is a naturally occurring compound and a potentirreversible inhibitor of SAH hydrolase. Deoxyl modification oferitadenine at the beta carbon results in a compound that is areversible inhibitor, while retaining inhibitory potency. Derivatives of4(adenine-9-yl)-2-hydroxybutanoic acid can be synthesized usingconventional synthetic methods known to one of ordinary skill in theart. (See e.g., Yuan et al., Adv. Antiviral Drug Des. 2: 41-88 (1996);Holy et al., Coll. Czechoslovak Chem. Commun. 50: 245-279 (1985)).

[0078] Examples of 4(adenine-9-yl)-2-hydroxybutanoic acids derivativesinclude, but are not limited to, base-modified derivatives, andside-chain substituted derivatives. Base modified derivatives arederivatives of 4(adenine-9yl)-2-hydroxybutanoic acids with modificationsat the adenyl ring base. The adenyl ring can be modified with variousmodifying groups at the amino group. The adenyl ring can also bemodified with various substituents at the C2 and C8 positions of theadenyl ring.

[0079] In one embodiment, the reversible inhibitors of SAH hydrolasehave the following formula (I), and pharmaceutically acceptable saltsthereof:

[0080] wherein Z is carbon or nitrogen, R1 and R2 are the same ordifferent, and are hydrogen, hydroxy, alkyl, cycloalkyl, alkenyl,alkoxy, amino, aryl, heteroaryl, or halogen; R3 and R4 are the same ordifferent and are hydrogen, alkyl, acetyl, alkenyl, aryl, or heteroaryl;X is oxygen, nitrogen, or sulfur; and Y is hydrogen, a C₁₋₁₀ alkylgroup, alkenyl, vinyl, aryl, or heteroaryl.

[0081] The different R groups can be optionally substituted with othersubstituents. These substituents may be halogen, hydroxy, alkoxy, nitro,cyano, carboxylic acid, alkyl, alkenyl, cycloalkyl, thiol, amino, acyl,carboxylate, aryl, carbamate, carboxamide, sulfonamide, a heterocyclicgroup, or any appropriate substituent known in the art. In a particularembodiment, each R group is hydrogen, or a lower straight chain alkylsuch as methyl. In another embodiment, one or more carbon atoms in thealkyl or alkoxy groups may be replaced by one or more heteroatoms.

[0082] The amino group may also be substituted once or twice to form asecondary or tertiary amine. Non-limiting examples of substituentsinclude alkyls or an optionally substituted alkyl group; alkene or anoptionally substituted alkenyl group; cycloalkyl or an optionallysubstituted cycloalkyl group; aryl, heterocyclic; aralkyl (e.g. phenylC₁₋₄ alkyl); heteroalkyl such as phenyl, pyridine, phenylmethyl,phenethyl, pyridinylmethyl, pyridinylethyl; and other substituents. Theheterocyclic group may be a 5 or 6 membered ring containing 1-4heteroatoms.

[0083] The amino group may be substituted with an optionally substitutedC₂₋₄ alkanoyl (e.g. acetyl, propionyl, butyryl, isobutyryl etc.); a C₁₋₄alkylsulfonyl (e.g. methanesulfonyl, ethanesulfonyl, etc.); a carbonylor sulfonyl substituted aromatic or heterocyclic ring (e.g.benzenesulfonyl, benzoyl, pyridinesulfonyl, pyridinecarbonyl etc.).

[0084] The CO—X—Y group can be an optionally substituted carboxylategroup. Examples of the optionally substituted carboxylate group include,but are not limited to, an optionally substituted alkyl (e.g. C₁₋₁₀alkyl); an optionally substituted cycloalkyl (e.g. C₃₋₇ cycloalkyl); anoptionally substituted alkenyl (e.g. C₂₋₁₀ alkenyl); an optionallysubstituted cycloalkenyl (e.g. C₃₋₇ cycloalkenyl); an optionallysubstituted aryl (e.g. phenyl, naphthyl, C₁₋₄ aryl such as benzyl); andother appropriate substituents. Groups such as methoxymethyl,methoxyethyl, and related groups are also encompassed.

[0085] Structure-based Drug Design of Novel SAH Hydrolase Inhibitors

[0086] It is also an object of the present invention to providestructure-based drug design using the compounds of the present inventionas an initial template molecule. Recently, X-ray structures of SAHhydrolase have become available for both “open” and “closed” forms ofthe enzyme. Using structure-based design, one of ordinary skill in theart can design novel compounds for screening SAH hydrolase inhibitors.The design or selection of candidate compounds can begin with theselection of various moieties which fill binding pockets of the SAHhydrolase. (See e.g., U.S. Pat. No. 5,756,466; Klebe, J. Mol. Med. 78:69-281 (2000); and Maignan et al., Curr. Top. Med. Chem. 1: 161-174(2001)).

[0087] There are a number of ways to select moieties to fill individualbinding pockets. These include visual inspection of a physical model orcomputer model of the active site and manual docking of models ofselected moieties into various binding pockets. Modeling software thatis well known and available in the art can be used. These include, butare not limited to, QUANTA (Molecular Simulations, Inc., Burlington,Mass., 1992); SYBYL (Molecular Modeling Software, Tripos Associates,Inc., St. Louis, Mo., 1992); AMBER (Weiner et al., J. Am. Chem. Soc. 6:765-784 (1984)); CHARMM (Brooks et al., J. Comp. Chem. 4: 187-217(1983)). The modeling step can be followed by energy minimization withstandard molecular mechanics forcefields such as CHARMM and AMBER. Inaddition, there are a number of more specialized computer programs toassist in the process of selecting the binding moieties of thisinvention. These include, but are not limited to:

[0088] 1. GRID (Goodford, “A Computational Procedure for DeterminingEnergetically Favorable Binding Sites on Biologically ImportantMacromolecules,”J. Med. Chem. 28: 849-857 (1985)). GRID is availablefrom Oxford University, Oxford, UK.

[0089] 2. MCSS (Miranker et al., “Functionality Maps of Binding Sites: AMultiple Copy Simultaneous Search Method,” in Proteins: Structure,Function and Genetics” 11: 29-34 (1991)). MCSS is available fromMolecular Simulations, Burlington, Mass.

[0090] 3. AUTODOCK (Goodsell et al., “Automated Docking of Substrates toProteins by Simulated Annealing,” in PROTEINS: Structure, Function andGenetics 8: 195-202 (1990)). AUTODOCK is available from the ScrippsResearch Institute, La Jolla, Calif.

[0091] 4. DOCK (Kuntz et al., “A Geometric Approach toMacromolecule-Ligand Interactions,” J. Mol. Biol. 161: 269-288 (1982)).DOCK is available from the University of California, San Francisco,Calif.

[0092] Once suitable binding moieties have been selected, they can beassembled into a single inhibitor. This assembly may be accomplished byconnecting the various moieties to a central scaffold. The assemblyprocess may, for example, be done by visual inspection followed bymanual model building, again using software such as QUANTA or SYBYL. Anumber of other programs may also be used to help select ways to connectthe various moieties. These include, but are not limited to:

[0093] 1. CAVEAT (Bartlett et al., “CAVEAT: A Program to Facilitate theStructure-Derived Design of Biologically Active Molecules,” in MolecularRecognition in Chemical and Biological Problems, Special Pub., RoyalChem. Soc. 78: 182-196 (1989)). CAVEAT is available from the Universityof California, Berkeley, Calif.

[0094] 2. 3D Database systems such as MACCS-3D (MDL Information Systems,San Leandro, Calif.). This area has been recently reviewed by Martin(Martin, “3D Database Searching in Drug Design,” J. Med. Chem. 35:2145-2154 (1992)).

[0095] 3. HOOK (available from Molecular Simulations, Burlington, Mass.)

[0096] In addition to the above computer assisted modeling of inhibitorcompounds, the inhibitors of this invention may be constructed de novousing either an empty active site or optionally including some portionsof a known inhibitor. Such methods are well known in the art. Theyinclude, for example:

[0097] 1. LUDI (Bohm, “The Computer Program LUDI: A New Method for theDe Novo Design of Enzyme Inhibitors,” J. Comp. Aid. Molee. Design 6:61-78 (1992)). LUDI is available from Biosym Technologies, San Diego,Calif.

[0098] 2. LEGEND (Nishibata et al., Tetrahedron, 47: 8985 (1991)).LEGEND is available from Molecular Simulations, Burlington, Mass.

[0099] 3. LeapFrog (available from Tripos associates, St. Louis, Mo.).

[0100] A number of techniques commonly used for modeling drugs may beemployed (see e.g., Cohen et al., J. Med. Chem. 33: 883-894 (1990)).Likewise a number of examples in the chemical literature of techniquescan be applied to specific drug design projects. (For a review, see,Navia et al., Curr. Opin. Struc. Biol. 2: 202-210 (1991)). Using thenovel combination of steps of the present invention, the skilled artisancan advantageously avoid time consuming and expensive experimentation todetermine enzymatic inhibition activity of particular compounds. Themethod is also useful in facilitating rational design of SAH hydrolaseinhibitors, and therapeutic and prophylactic agents against SAHhydrolase-mediated diseases. Accordingly, the present invention relatesto such inhibitors, and methods for identifying or selecting suchinhibitors.

[0101] A variety of conventional techniques may be used to carry outeach of the above evaluations, as well as evaluations necessary inscreening a candidate compound for SAH hydrolase inhibiting activity.Generally, these techniques involve determining the location and bindingproximity of a given moiety, the occupied space of a bound inhibitor,the deformation energy of binding of a given compound and electrostaticinteraction energies. Examples of conventional techniques useful in theabove evaluations include, but are not limited to, quantum mechanics,molecular mechanics, molecular dynamics, Monte Carlo sampling,systematic searches and distance geometry methods (Marshall, Ann. Ref.Pharmacol. Toxicol. 27: 193 (1987)). Specific computer software has beendeveloped for use in carrying out these methods. Examples of programsdesigned for such uses include: Gaussian 92 (Gaussian, Inc., Pittsburgh,Pa.); AMBER; QUANTA/CHARMM; and Insight II/Discover (BiosysmTechnologies Inc., San Diego, Calif.). These programs may beimplemented, for instance, using a Silicon Graphics Indigo2 workstationor IBM RISC/6000 workstation model 550. Other hardware systems andsoftware packages will be known and be of evident applicability to thoseskilled in the art.

[0102] Different classes of active SAH hydrolase inhibitors, accordingto this invention, may interact in similar ways with the various bindingpockets of the SAH hydrolase active site. The spatial arrangement ofthese important groups is often referred to as a pharmacophore. Theconcept of the pharmacophore has been well described in the literature(See Mayer et al., J. Comp. Aided Molec. Design 1: 3-16 (1987);Hopfinger et al. in Concepts and Applications of Molecular Similarity,Johnson and Maggiora (eds.), Wiley (1990))

[0103] Different classes of SAH hydrolase inhibitors of this inventionmay also use different scaffolds or core structures that allow thenecessary moieties to be placed in the active site such that thespecific interactions necessary for binding may be obtained. Thesecompounds are best defined in terms of their ability to match thepharmacophore, i.e., their structural identity relative to the shape andproperties of the active site of SAH hydrolase. Various scaffolds havebeen described in, for example, Klebe, G., J. Mol. Med. 78: 269-281(2000); Maignan et al., Curr. Top. Med. Chem. 1: 161-174 (2001); andU.S. Pat. No. 5,756,466 to Bemis et al.).

[0104] S-Adenosyl-L-Homocysteine Hydrolase To Be Inhibited

[0105] The compounds of the present invention can be used to reversiblyinhibit any SAH hydrolase. It is not intended that the present inventionbe limited to reversibly inhibiting any specific SAH hydrolase.

[0106] In one embodiment, the compounds of the present invention can beused to reversibly inhibit SAH hydrolase encoded by nucleic acidscontaining nucleotide sequences with the following GenBank accessionNos.: AF129871 (Gossypium hirsutum); AQ003753 (Cryptosporidium parvum);AF105295 (Alexandrium fundyense); AA955402 (Rattus norvegicus); AA900229(Rattus norvegicus); AA874914 (Rattus norvegicus); AA695679 (Drosophilamelanogaster ovary); AA803942 (Drosophila melanogaster ovary; All 87655(Manduca sexta male antennae); U40872 (Trichomonas vaginalis); AJ007835(Xenopus Laevis); AF080546 (Anopheles gambiae); A1069796 (T. cruziepimastigote); Z97059 (Arabidopsis thaliana); AF059581 (Arabidopsisthaliana); U82761 (Homo sapiens); AA754430 (Oryza sativa); D49804(Nicotiana tabacum); D45204 (Nicotiana tabacum); X95636 (D.melanogaster); T18277 (endosperm Zea mays); R75259 (Mouse brain); Z26881(C. roseus); X12523 (D. discoideum); X64391 (Streptomyces fradiae);W21772 (Maize Leaf); AH003443 (Rattus norvegicus); U14963 (Rattusnorvegicus); U14962 (Rattus norvegicus); U14961 (Rattus norvegicus);U14960 (Rattus norvegicus); U14959 (Rattus norvegicus); U14937 (Rattusnorvegicus); U14988 (Rattus norvegicus); U14987 (Rattus norvegicus);U14986 (Rattus norvegicus); U14985 (Rattus norvegicus); U14984 (Rattusnorvegicus); U14983 (Rattus norvegicus); U14982 (Rattus norvegicus);U14981 (Rattus norvegicus); U14980 (Rattus norvegicus); U14979 (Rattusnorvegicus); U14978 (Rattus norvegicus); U14977 (Rattus norvegicus);U14976 (Rattus norvegicus); U14975 (Rattus norvegicus); L32836 (Musmusculus); L35559 (Xenopus laevis); Z19779 (Human foetal Adrenalstissue); L23836 (Rhodobacter capsulatus); M15185 (Rat); L11872 (Triticumaestivum); M19937 (Slime mold (D. discoideum); M80630 (Rhodobactercapsulatus).

[0107] In another embodiment, the compounds of the present invention canbe used to reversibly inhibit SAH hydrolase encoded by nucleic acidscontaining nucleotide sequences with the GenBank accession Nos.M61831-62832 (see also Coulter-Karis and Hershfield, Ann. Hum. Genet.,53(2):169-175 (1989)). The compounds of the present invention can alsobe used to reversibly inhibit SAH hydrolase encoded by nucleic acidscontaining the nucleotide or amino acid sequences set forth in U.S. Pat.No. 5,854,023.

[0108] C. Use as Therapeutic Agents

[0109] Reversible inhibition of SAH hydrolase using4(adenine-9-yl)-2-hydroxybutanoic acid, its derivatives, andpharmaceutically acceptable salts results in significantly reducedcytotoxicity while retaining its therapeutic effects. With its potencyand reversibility, the compounds of the present invention can be used astherapeutic agents without the severe toxicity associated with otherirreversible inhibitors. The compounds of the present invention areuseful as agents demonstrating biological activities related to theirability to inhibit SAH hydrolase. The inhibitory effect on SAH hydrolasecan be evaluated using the ratio of the initial rates of SAH hydrolysisin the presence or absence of the inhibitor, or using any methods knownto one of ordinary skill in the art. The present invention providescompositions and methods' for the prevention and treatment of diseasessuch as hemorrhagic viral infection, autoimmune disease, autograftrejection, neoplasm, hyperhomocysteineuria, cardiovascular disease,stroke, Alzheimer's disease, and diabetes. However, it is not intendedthat the present invention be limited to the prevention and treatment ofparticular diseases.

[0110] 1. Hemorrhagic Fever Viruses

[0111] The present invention provides compositions and methods for thetreatment of viral hemorrhagic fever. The reversible inhibitors of thepresent invention can serve as a broad-spectrum antiviral agent againstall types of viruses causing hemorrhagic fever, including, but notlimited to, togavirus, arenavirus, nairovirus, and hantavirus.Broad-spectrum antiviral drugs offer many advantages overnarrow-spectrum agents. Because of the difficulty associated withclinical diagnoses of viral pathogens, diagnostic results often arrivetoo late for the choice of a specific antiviral drug. Immediate actionis often necessary to prevent the condition of the patient fromworsening, particularly in acute infections where viral chemotherapymust start as soon as the patient presents clinical symptoms.

[0112] Inhibitors of S-adenosyl-L-homocysteine (SAH) hydrolase have beenreported to be effective in the treatment of Ebola viral infections. Thecompounds of the present invention can also be used against otherhemorrhagic diseases, such as those described in WO 00/64479. Althoughthe mechanism of inhibition is not necessary in practicing the methodsof the present invention, the mechanism of action by which the compoundsof the present invention inhibit viral replication may be based oninhibition of viral methylation.

[0113] 2. Autoimmune Diseases and Diseases Associated WithImmunosuppression

[0114] The present invention contemplates compositions and methods forpreventing and treating autoimmune diseases. If a person has anautoimmune disease, the immune system mistakenly attacks the cells,tissues, and organs of a person's own body. As a group, autoimmunediseases afflict millions of Americans. Most autoimmune diseases strikewomen more often than men. Examples of autoimmune diseases can be foundfrom the National Institute of Health, “Understanding AutoimmuneDisease”(http://www.niaid.nih.gov/publications/autoimmune/autoimmune.htm.).

[0115] Compounds that modulate SAH hydrolase activity may also be usedfor the treatment of diseases that are associated withimmunosuppression. Immunosuppression can be due to chemotherapy,radiation therapy, enhanced wound healing, enhanced burn treatment, orother drug therapy such as corticosteroid therapy, or a combination ofdrugs used in the treatment of autoimmune diseases andgraft/transplantation rejection. Immunosuppression can also be due tocongenital deficiency in receptor function, infectious diseases,parasitic diseases, or other causes.

[0116] 3. Neoplasm and Cancer

[0117] The present invention also contemplates compositions and methodsfor preventing and treating neoplasms, including, but not limited toneoplasm associated with the adrenal gland, anus, auditory nerve, bileducts, bladder, bone, brain, breast, bruccal, central nervous system,cervix, colon, ear, endometrium, esophagus, eye, eyelids, fallopiantube, gastrointestinal tract, head, neck, heart, kidney, larynx, liver,lung, mandible, mandibular condyle, maxilla, mouth, nasopharynx, nose,oral cavity, ovary, pancreas, parotid gland, penis, pinna, pituitary,prostate gland, rectum, retina, salivary glands, skin, small intestine,spinal cord, stomach, testes, thyroid, tonsil, urethra, uterus, vagina,vestibulocochlear nerve, vulva, and neoplasm associated with otherorgans. In particular embodiments, the pharmaceutical compositions ofthe present invention are useful for the treatment of non-small celllung cancer, lung cancer, breast cancer, and prostate cancer. Thepresent invention further contemplates compositions and methods forpreventing and treating cancers, including, but not limited to thoseassociated with solid tumors, lymphoma, metastatic tumors, glioblastomatumors, and other carcinomas tumors.

[0118] 4. Diseases Associated With Increased Homocysteine Levels

[0119] Furthermore, it is contemplated that the compounds of the presentinvention can be used as a plasma homocysteine lowering agent for theprevention and treatment of diseases associated with increased levels ofhomocysteine. Diseases which have been found to be linked with increasedhomocysteine levels (i.e., hyperhomocysteinemia) include, but are notlimited to cardiovascular diseases, stroke, Alzheimer's disease anddiabetes. For example, various studies have shown a relation betweenhyperhomocysteinemia and coronary heart disease (CHD), peripheralvascular disease, stroke, and venous thrombosis.

[0120] The increased risk of stroke from high homocysteine levels alsoincrease the chance of developing Alzheimer's disease. Recent studieshave also shown that people with dementia of the Alzheimer's type haveelevated levels of homocysteine in their blood. (Selhub et al., “Plasmahomocysteine as a risk factor for dementia and Alzheimer's disease,” N.Eng. J. Med. 46: 476-483 (2002)). Elevated homocysteine has also beenlinked to complications in diabetes, lupus, and other chronic diseases.

[0121] D. Pharmaceutical Compositions

[0122] Pharmaceutical compositions of the present invention comprise anyof the compounds of the present invention and pharmaceuticallyacceptable salts thereof, alone or in combination with anypharmaceutically acceptable carriers, adjuvant or vehicle. Acceptablecompositions and methods for their administration that can be employedfor use in this invention include, but are not limited to thosedescribed in U.S. Pat. Nos. 5,736,154; 6,197,801; 5,741,511; 5,886,039;5,941,868; 6,258,374 and 5,686,102. Examples of pharmaceuticallyacceptable carriers, adjuvants and vehicles that can be used in thepharmaceutical compositions of this invention include, but are notlimited to, ion exchangers, alumina, aluminum stearate, lecithin, serumproteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

[0123] The formulation, dosage and route of administration can bedetermined according to methods known in the art (see e.g., Remington:The Science and Practice of Pharmacy, Alfonso R. Gennaro (Editor) MackPublishing Company, April 1997; Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems, Banga, 1999; andPharmaceutical Formulation Development of peptides and Proteins,Hovgaard and Frkjr (Ed.), Taylor & Francis, Inc., 2000;Biopharmaceutical Drug Design and Development, Wu-Pong and Rojanasakul(Ed.), Humana Press, 1999). In the treatment or prevention of conditionswhich require SAH hydrolase modulation, an appropriate dosage level willgenerally be about 0.01 to 500 mg per kg body weight per day.Preferably, the dosage level will be about 0.1 to about 250 mg/kg perday. In more preferred embodiments, the dosage level will range fromabout 0.1 to about 20 mg/kg per day. The appropriate dosage can beadministered in single or multiple dose. It will be understood that thespecific dose level and frequency of dosage for any particular subjectmay be varied and will depend upon a variety of factors, including theactivity of the specific compound used, the metabolic stability andlength of action of that compound, the age, body weight, general health,sex, diet, mode and time of administration, rate of excretion, drugcombination, the severity of the particular condition, and the patientundergoing therapy.

[0124] The pharmaceutical compositions of this invention can beadministered orally, parenterally, by inhalation spray, topically,rectally, nasally, buccally, vaginally, via an implanted reservoir, orany suitable form of administration. The term parenteral as used hereinincludes subcutaneous, intracutaneous, intravenous, intramuscular,intra-articular, intrasynovial, intrasternal, intrathecal, intralesionaland intracranial injection or infusion techniques. The most suitableroute in any given case will depend on the nature and severity of thecondition being treated and on the nature of SAH hydrolase inhibitorbeing used.

[0125] The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(e.g., Tween 80), and suspending agents. The sterile injectablepreparation may also be a sterile injectable solution or suspension in anon-toxic parenterally-acceptable diluent or solvent. For example, thepharmaceutical composition may be a solution in 1,3-butanediol. Otherexamples of acceptable vehicles and solvents that may be employed in thecompositions of the present invention include, but are not limited to,mannitol, water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or diglycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant.

[0126] The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, and aqueous suspensions and solutions. Inthe case of tablets for oral use, commonly used carriers include, butare not limited to, lactose and corn starch. Lubricating agents, such asmagnesium stearate, can also be added. For oral administration in acapsule form, useful diluents include lactose and dried corn starch.When aqueous suspensions are administered orally, the active ingredientis combined with emulsifying and suspending agents. If desired, certainsweetening, flavoring, and coloring agents may be added.

[0127] The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisinvention with a suitable non-irritating excipient. In particularembodiments, the excipient is solid at room temperature but liquid atthe rectal temperature. Thus, the excipient will melt in the rectum torelease the active components. Such materials include, but are notlimited to, cocoa butter, beeswax and polyethylene glycols.

[0128] The pharmaceutical compositions of this invention may beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation. For example, such composition may be prepared as solutionsin saline, employing benzyl alcohol or other suitable preservatives,absorption promoters to enhance bioavailability, fluorocarbons, and/orother solubilizing or dispersing agents known in the art.

[0129] The pharmaceutical compositions of this invention may also beadministered topically. For topical application to the skin, thepharmaceutical composition may be formulated with a suitable ointmentcontaining the active components suspended or dissolved in a carrier.Carriers for topical administration of the compounds of this inventioninclude, but are not limited to, mineral oil, liquid petroleum, whitepetroleum, propylene glycol, polyoxyethylene polyoxypropylene compound,emulsifying wax and water. Alternatively, the pharmaceutical compositioncan be formulated with a suitable lotion or cream containing the activecompound suspended or dissolved in a carrier. Suitable carriers include,but are not limited to, mineral oil, sorbitan monostearate, polysorbate60, cetyl esters wax, cetaryl alcohol, 2-octyldodecanol, benzyl alcoholand water. The pharmaceutical compositions of this invention may also betopically applied to the lower intestinal tract by rectal suppositoryformulation or in a suitable enema formulation. Topically-transdermalpatches are also included in this invention.

[0130] The invention also provides kits for carrying out the therapeuticregimens of the invention. Such kits comprise therapeutically effectiveamounts of an SAH hydrolase inhibitor, alone or in combination withother agents, in pharmaceutically acceptable form. Preferredpharmaceutical forms include inhibitors in combination with sterilesaline, dextrose solution, buffered solution, or other pharmaceuticallyacceptable sterile fluid. Alternatively, the composition may belyophilized or desiccated. In this instance, the kit may furthercomprise a pharmaceutically acceptable solution, preferably sterile, toform a solution for injection purposes. In another embodiment, the kitmay further comprise a needle or syringe, preferably packaged in sterileform, for injecting the composition. In other embodiments, the kitfurther comprises an instruction means for administering the compositionto a subject. The instruction means can be a written insert, anaudiotape, an audiovisual tape, or any other means of instructing theadministration of the composition to a subject.

[0131] E. Combinations for Reversibly Inhibiting SAH Hydrolase Activity

[0132] The present invention also provides combinations and kits forreversibly inhibiting SAH hydrolase activity. In one embodiment, thepresent invention provides a combination, comprising an effective amountof a compound having formula I; and an effective amount of ananti-hemorrhagic viral infection agent, an immunosuppressant, a plasmahomocysteine lowering agent, or an anti-neoplasm agent. The combinationcan further comprise a pharmaceutically acceptable carrier or excipient.In yet another aspect, the present invention provides a kit, comprisingan effective amount of the combination as described, and an instructionmeans for administering the combination to a subject.

[0133] Any agent that can alleviate or ameliorate clinical symptoms ordiagnostic markers associated with viral hemorrhagic diseases can beused in the combination of the present invention. Anti-viral therapeuticagents include, but are not limited to, anti-viral vaccines, anti-viralantibiotics, viral-activated immune cells and viral-activated immunesera. WO 00/64479 describes examples of anti-viral therapeutic agentsthat can be used in the combination of the present invention. Preferredembodiments are antiviral therapeutic agents that exhibit biologicalactivity against viral hemorrhagic diseases caused by infection of aBunyaviridaea, a Filoviridae, a Flaviviridae, or an Arenaviridae virus.

[0134] Any agent that suppresses the ability of the body's immune systemto fight disease can be used in the combination of the presentinvention. Non-limiting examples of immunosuppressants are cyclosporine,prednisilone, azathioprine, tacrolimus, an adrenocortical steroid,mycophenolate, cyclophosphamide, methotrexate, chlorambucil,vincristine, vinblastine, dactinomycin, an antithymocyte globulin,muromonab-CD3 monoclonal antibody, Rh₀(D) immune globulin, methoxsalen,and thalidomide (See, Goodman & Gilman's The Pharmacological Basis ofTherapeutics, (9th Ed.) McGraw-Hill 1996, pages 1294-1304). Theimmunosuppressant can be taken as a combination of drugs. For example,most people start on a combination of drugs (e.g., cyclosporin,azathioprine, and prednisilone combination) after their transplant. Overa period of time, the doses of each drug and the number of drugs takenmay be reduced as the risks of rejection decline.

[0135] Any agent that lowers homocysteine levels can be used in thecombination of the present invention. Folic acid is known to be aneffective homocysteine-lowering agent. Other homocysteine-loweringagents include, but are not limited to, betaine, trimethylglycin,cyanobalamin, and other B-group vitamins. The combination can alsoinclude any multi-vitamin and mineral supplement for use in loweringhomocysteine. Examples of multivitamin and mineral supplements that canbe used in the combinations of the present invention, include, but arenot limited to, those described in U.S. Pat. Nos. 6,361,800; 6,353,003;6,323,188; 6,274,170; 6,210,686; 6,203,818; and 5,668,173.

[0136] Any anti-neoplasm agent can be used in the combination of thepresent invention. Examples of anti-neoplasm agents that can be used inthe compositions and methods of the present invention are described inU.S. patent application No. 2002/044919. In one embodiment, theanti-neoplasm agent used is an anti-angiogenic agent. Theanti-angiogenic agent can be an inhibitor of basement membranedegradation, an inhibitor of cell migration, an inhibitor of endothelialcell proliferation, and an inhibitor of three-dimensional organizationand establishment of potency. Examples of such anti-angiogenic agent areillustrated in Auerbach and Auerbach, Pharmacol. Ther., 63: 265-311(1994); O'Reilly, Investigational New Drugs, 15: 5-13 (1997); J. Nat'lCancer Instit., 88: 786-788 (1996); and U.S. Pat. Nos. 5,593,990;5,629,327 and 5,712,291. In another embodiment, the anti-neoplasm agentused is an alkylating agent, an antimetabolite, a natural product, aplatinum coordination complex, an anthracenedione, a substituted urea, amethylhydrazine derivative, an adrenocortical suppressant, a hormone,and an antagonist.

[0137] Other anti-neoplasm agents include, but are not limited to,cytidine, arabinosyladenine (araC), daunomycin, doxorubicin,methotrexate (MTX), fluorinated pyrimidines such as 5-fluorouracil(5-FU), hydroxyurea, 6-mercaptopurine, plant alkaloids such asvincristine (VCR), VP-16 and vinblastine (VLB), alkylating agent,cisplatin, nitrogen Mustard, trisamine, procarbazine, bleomycin,mitomycin C, actinomycin D, or an enzyme such as L-Asparaginase. Theanti-neoplasm agent can also be an oncogene inhibitor such as ananti-oncogene antibody or an anti-oncogene antisense oligonucleotide. Inanother embodiment, the anti-neoplastic agent is a cellular matrixinhibitor such as an anti-cellular-matrix antibody or ananti-cellular-matrix antisense oligonucleotide. For example, antibodiesand antisense oligonucleotides against caveolin-1, decorin, cadherins,catenins, integrins, and other cellular matrix or cellular matrix genescan be used.

[0138] In a specific embodiment, the combination further comprises atumor suppressor gene for combined intratumoral therapy and genetherapy. The gene can be used in the form of naked DNA, complexed DNA,cDNA, plasmid DNA, RNA or other mixtures thereof as components of thegene delivery system. In another embodiment, the tumor suppressor geneis included in a viral vector. Any viral vectors that are suitable forgene therapy can used in the combination. For example, an adenovirusvector (U.S. Pat. No. 5,869,305), a simian virus vector (U.S. Pat. No.5,962,274), a conditionally replicating human immunodeficiency viralvector (U.S. Pat. No. 5,888,767), retrovirus, SV40, Herpes simplex viralamplicon vectors and vaccinia virus vectors can be used. In addition,the genes can be delivered in a non-viral vector system such as aliposome wherein the lipid protects the DNA or other biomaterials fromoxidation during the coagulation.

F. EXAMPLES Example 1 Synthesis of Reversible Inhibitors of SAHHydrolase

[0139]¹H (Me₄Si) NMR spectra were determined with solution in CDCl₃ at400 MHz, ¹³C (Me₄Si) at 100.6 MHz unless otherwise noted. Mass spectra(MS) were obtained by atmospheric pressure chemical ionization (APCI)technique. Reagent grade chemicals were used. Solvents were dried byreflux over and distillation from CaH₂ under an argon atmosphere, exceptTHF, which was distilled from benzophenone and potassium. TLC wasperformed on Merck kieselgel 60-F₂₅₄ with MeOH/CHCl₃ (1:9) andEtOAc/MeOH (95:5) as developing systems, and products were detected with254 nm light. Merck kieselgel 60 (230-400 mesh) was used for columnchromatography.

[0140] Elemental analyses were determined by Galbraith Laboratories,Knoxville, Tenn. Spectral data for isolated compounds were consistentwith reported data. (Holy, Coll. Czech. Chem. Commun. 43, 3444-3464(1978); Holy et al., Coll. Czech. Chem. Commun. 50: 262-279 (1985);Japanese Patent 69-50781; Chem. Abstr. 1972: 514811). The syntheses areschematically shown in Scheme 2.

[0141] 9-(3,4-O-Isopropylidene-3,4-dihydroxybutyl)adenine (1)

[0142]¹H NMR δ 1.36 (s, 3, CH₃), 1.45 (s, 3, CH₃), 2.00-2.05 (m, 1,H2′), 2.22-2.24 (m, 1, H2″), 3.57-3.59 (m, 1, H3′), 4.03-4.06 (m, 2,H4′,4″), 4.34-4.43 m, 2, H1′, 1″), 5.76 (br s, 2, NH₂), 7.86 (s, 1, H8),8.38 (s, 1, H2); MS (APCI) m/z 264 (100, MH⁺).

[0143] 9-(3,4-Dihydroxybutyl)adenine (2)

[0144] A solution of 1 (110 mg, 0.18 mmol) in CF₃COOH/H₂O (9:1) (5 ml)was stirred for 20 min at ˜0° C. Volatiles were evaporated, coevaporatedwith toluene (3×) and EtOH (2×) to give 2 (73 mg, 78%) aftercrystallization from EtOH with spectra data as reported.

[0145] 9-(4-O-t-Butyldimethylsilyl-3,4-dihydroxybutyl)adenine (3)

[0146] TBDMS-Cl (186 mg, 1.23 mmol) and imidazole (168 mg, 2.46 mmol)were added to a stirred solution of 2 (250 mg, 1.12 mmol) in dry DMF (8mL). The mixture was stirred at ambient temperature for 5 h, thenreaction mixture was partitioned between EtOAc/NH₄Cl/H₂O. The waterlayer was extracted with next portion of EtOAc. The combined organicphase was washed (brine), dried (Na₂SO₄), evaporated and the residue wascolumn chromographed (CHCl₃/MeOH; 97: 3) to give 3 (234 mg, 62%): ¹H NMRδ 0.04 (s, 6, 2×CH₃), 0.88 (s, 9, t-Bu), 1.81-1.89 (m, 1, H2′),2.03-2.11 (m, 1, H2″) 3.50-3.52 (m, 2, H4′,4″), 3.58-3.59 (m, 1, H3′),4.32-4.45 (m, 2, H1′,1″), 6.10 (br s, 2, NH₂), 7.87 (s, 1, H8), 8.36 (s,1, H2); ¹³C NMR δ −5.0 & −4.9 (2×CH ₃) 18.7 (t-Bu), 26.3 (t-Bu), 33.7(C2′), 40.9 (C 1′), 67.4 (C4′), 68.4 (C3′), 119.9 (C5), 141.4 (C8),150.5 (C4), 153.1 (C2), 155.8 (C6); MS (APCI) m/z 338 (100, MH⁺). Anal.Calcd for C₁₅H₂₇N₅O₂Si (337.50): C, 53.38; H, 8.06; N, 20.75.

[0147] 9-[4-O-t-Butyldimethylsilyl-3-O-(1-ethoxyethyl-3,4-dihydroxybutyl] adenine (4)

[0148] Ethyl vinyl ether (214 mg, 0.28 mL, 2.96 mmol) and pyridiniump-toluenesulfonate (15 mg, mmol) were added to a solution of 3 (250 mg,0.74 mmol) in dry CH₂Cl₂ (30 mL), and the mixture was stirred at ambienttemperature under N₂ until no starting material was detected by TLC(usually 5-6 days). Then reaction mixture was washed with water, dried(Na₂SO₄), and was evaporated. Column chromatography (EtOAc/MeOH; 97: 3)gave 4 (160 mg, 53%) as 1:1 mixture of diastereoisomers: ¹H NMR δ 0.04(s, 6,2×CH₃), 0.88 (s, 9, t-Bu), 1.18-1.34 (complex m, 6, 2×CH₃),2.05-2.21 (m, 2, H2′,2″), 3.50-3.63 (complex m, 4, H4′,4″, CH₂),3.70-3.82 (m, 1, H3′), 4.31-4.40 (m, 2, H1′, 1″), 4.72-4.78 & 4.88-4.93(2×m, 1, CH), 5.99 & 6.06 (2×br s, 2, NH₂), 7.86 & 8.04 (2×s, 1, H8),8.37 (s, 1, H2); ³C NMR 6-5.1(2×CH₃), 15.7 & 15.9 (CH₃), 18.6 (t-Bu),20.7 & 21.0 (CH₃), 26.2 (t-Bu), 32.5 & 32.7 (C2′), 41.0 & 41.1 (C1′),61.0 & 62.1 (CH₂), 65.5 & 66.1 (C4′), 73.6 & 74.8 (C3′), 100.2 & 100.5(CH), 119.9 (C5), 141.1 & 142.2 (C8), 150.5 (C4), 152.1 & 152.5 (C2),155.3 (C6); MS (APCI) m/z 410 (100, MH⁺). Anal. Calcd for C₁₉H₃₅N₅O₃Si(409.60): C, 55.71; H, 8.61; N, 17.10.

[0149] 9-[3-O-(1-Ethoxyethyl)-3,4-dihydroxybutyl]adenine (5)—Procedure A

[0150] TBAF/THF (0.88 mL, IM) was added to a solution of 4 (180 mg, 0.44mmol) in dry THF (6 mL) and the mixture was stirred at ambienttemperature for 20 min. Volatiles were evaporated and the residue wascolumn chromatographed (EtOAc/MeOH; 78:12) to give 5 (120 mg, 92%) as1:1 mixture of diastereoisomers: ¹H NMR δ 1.21-1.25 (complex m, 3, CH₃),1.35 & 1.39 (d, J=5.3 Hz, 3, CH₃), 2.05-2.25 (m, 2, H2′,2″), 3.53-3.82(complex m, 5, H3′,4′,4″, CH₂), 4.32-4.39 (m, 2, H1,1″), 4.69 &4.88-4.87 (q, J=5.3 Hz, 1, CH), 5.88 & 5.92 (2×br s, 2, NH₂), 7.82 &7.94 (2×s, 1, H8), 8.36 (s, 1, H2); ¹³C NMR δ 15.5 & 15.7 (CH₃), 20.4 &20.6 (CH₃), 32.5 & 32.7 (C2′), 40.0 & 41.1 (C1′), 60.9 & 62.4 (CH₂),64.9 & 65.9 (C4′), 73.2 & 78.9 (C3′), 99.6 & 101.5 (CH), 119.7 (C5),140.8 & 141.5 (C8), 150.5 (C4), 152.8 & 153.1 (C2), 155.6 & 155.7 (C6);MS (APCI) m/z 296 (100, MH⁺). Anal. Calcd for C₁₃H₂₁N₅O₃ (295.34): C,52.87; H, 7.17; N, 23.71.

[0151] Methyl 4-(Adenin-9-yl)-2-hydroxybutanoate (6)—Procedure B

[0152] To a suspension of 5 (90 mg, 0.31 mmol) in CH₃CN/CCl₄/H₂O(1:1:1.5; 1.5 mL), NaHCO₃ (161 mg, 0.88 mmol), NaIO₄ (353 mg, 1.65 mmol)and RuCl₃ (trace) were added. The mixture was stirred at ambienttemperature for 48 h until no starting material was detected on TLC.Then water (5 mL) and CHCl₃ (4 mL) were added, the two layers wereseparated and water phase was washed with CHCl₃ (3 mL). The aqueouslayer was acidified with HCl to pH 4 and applied on a column of Dowex50W×2 (H⁺). Column was washed with 200 mL of water then product waseluted with 2.5% NH₄OH/H₂O. The combined UV-absorbing ammonia eluate wasevaporated and coevaporated with MeOH (2×). The residue was dissolved inMeOH (5 mL) and a solution of CH₂N₂ in diethyl ether was added untilyellow color of diazomethane was maintained during several minutes. Thesolution was concentrated and column chromatographed (CHCl₃/MeOH; 95:5)to give 6 (31 mg, 41%) as a white solid with data identical as reported.To avoid formation of by-product 6′ it is imported to keep desiredamount of NaHCO₃ in reaction mixture.

[0153] 4-(Adenin-9-yl)-2-hydroxybutanoic acid (7)—Procedure C

[0154] NaOH/H₂O (1 mL. 0.1 M) was added to a solution of 6 (10 mg, 0.04mmol) in MeOH/H₂O (2.0 mL). The mixture was stirred at ambienttemperature for 6 h until no starting material was detected on TLC. Thenreaction mixture was acidified with HCl to pH ˜4 and applied on a columnof Dowex 50W×2 (H⁺). Column was washed with water (100 mL) and thenproduct was eluted with 2.5% NH₄OH. The combined UV- absorbing ammoniaeluate was evaporated to give 7 as a ammonium salt (7.6 mg, 75%) withdata as reported.

[0155] 9-(3,4-O-Di-t-Butyldimethylsilyl-3,4-dihydroxybutyl)adenine (8)

[0156] TBDMS-Cl (593 mg, 3.92 mmol) and imidazole (534 mg, 7.85 mmol)were added to a stirred solution of 2 (350 mg, 1.57 mmol) in dry DMF (8mL), and the mixture was stirred at ambient temperature overnight. Thenreaction mixture was partitioned between EtOAc/NH₄Cl/H₂O. The waterlayer was extracted with EtOAc. The combined organic phase was washed(brine), dried (Na₂SO₄), and evaporated. Column chromatography (CHCl₃ 3%MeOH/CHCl₃) gave 8 (610 mg, 86%): ¹H NMR δ 0.04 (s, 6, 2×CH₃), 0.09 (s,6, 2×CH₃), 0.88 (s, 9, t-Bu), 0.92 (s, 9, t-Bu), 2.02-2.07 (m, 1, H2′),2.21-2.26 (m, 1, H2″), 3.46 (dd, J=6.8, 10.0 Hz, 1, H4′), 3.59 (dd,J=5.2, 10.0 Hz, 1, H4″), 3.78-3.81 (m, 1, H3′), 4.32-4.45 (m, 2,H1′,1″), 5.96 (br s, 2, NH₂), 7.84 (s 1, H8), 8.42 (s, 1, H2); MS (APCI)m/z 452 (100, MH⁺). Anal. Calcd for C₂₁H₄₁N₅O₂Si₂ (451.76): C, 55.83; H,9.15; N, 15.50.

[0157] 9-(3-O-t-Butyldimethylsilyl-3,4-dihydroxybutyl)adenine (9)

[0158] Compound 8 (400 mg, 0.887 mmol) was added to a solution ofCH₃CO₂H/H₂O/THF (13:7:3; 8 mL) and the mixture was stirred at ambienttemperature until more polar spot of compound 2 starting to appear onTLC. Then reaction mixture was partitioned (EtOAc//NaHCO₃/H₂O) and theaqueous layer was extracted with next portion of EtOAc. The combinedorganic phase was washed (NaHCO₃, brine), dried (Na₂SO₄), evaporated andcolumn chromatographed (CHCl₃ →4% MeOH/CHCl₃) to give recovered 8 (140mg, 35%) and 9(155 mg, 52%): ¹HNMR δ 0.04(s, 6, 2×CH₃), 0.88 (s, 9,t-Bu), 2.16-2.19 (m, 2, H2′,2″), 3.61 (dd, J=4.5, 11.4 Hz, 1, H4″), 3.65(dd, J=5.2, 11.4 Hz, 1, H4″), 3.88 (q, J=5.2 Hz, 1, H3′), 4.26-4.36 (m,2, H1′,1″), 6.17 (br s, 2, NH₂), 7.80 (s, 1, H8), 8.29 (s, 1, H2); ¹³CNMR δ −4.4 & 4.1 (CH₃), 18.5 (t-Bu), 26.2 (t-Bu), 34.5 (C2′), 40.8(C1′), 65.5 (C4′), 70.6 (C3′), 119.8 (C5), 140.7 (C8), 150.4 (C4), 153.0(C2), 155.5 (C6); MS (APCI) m/z 338 (100, MH⁺). Anal. Calcd. forC₁₅H₂₇N₅O₂Si (337.50): C, 53.38; H, 8.06; N, 20.75.

[0159] Methyl 3-(Adenin-9-yl)propionate (6′)

[0160] Treatment of 9 (50 mg, 0.148 mmol) by procedure B (columnchromatography: CHCl₃/MeOH 97:3) gave 6′ (9 mg, 27%) with data asreported.

[0161] 3-(Adenin-9-yl)propionic acid (7′)

[0162] Treatment of 6′ (10 mg, 0.045 mmol) by procedure C (columnchromatography: CHCl₃/MeOH 97:3) gave 6′ (7.3 mg, 78%) with data asreported.

Example 2 DZ2002 Mediates Immunosuppressive Effects

[0163] Materials and Methods

[0164] Reagents

[0165] AdoHcy hydrolase inhibitors DZ2002 was synthesized at DiazymeLaboratories. Con A (Concanavalin A), LPS (Escherichia coli 055:B5) andSac (Staphylococcus aureus Cowan strain 1) were obtained from Pansorbin®cells, Biosciences, inc.(La Jolla., CA 92039, USA). RPMI 1640 and fetalbovine serum (FBS) were obtained from GIBCO. Purified rat anti-mouseIL-10, IL-12p70, IL-12p40, IFN-γ and biotinylated anti-mouse IL-10,IL-12p70, IL-12p40, IFN-γ, FITC-anti-mouse-CD11b (Mac-1), Phycorythrin(PE)-anti-mouse I-Ad, PE-anti-human-CD14, PE- anti-human-ABC, PE-anti-human-DR, PE-anti-human-CD80 and PE-anti-human-CD86 were Pharmingenproducts. Thioglycollate (TG) is available from Sigma-Aldrich.

[0166] Animal

[0167] Inbred BALB/C mice, 6˜8 weeks of age, were provided by ShanghaiExperimental Animal Center of Chinese Academy of Sciences withCertificate No. 99-003. The mice were housed in specific pathogen-free(SPF) conditions with room temperature of 24±2° C., 12 hr light/darkcycle, and provided with sterile food and water ad libitum.

[0168] Cells

[0169] Spleens from Balb/c mice were aseptically removed, pooled, andsingle cell suspensions prepared in PBS. Erythrocytes were lysed bytreatment with Tris-buffered ammonium chloride (0.155 M NH₄CL, 0.0165 MTris, PH 7.2). Mononuclear cells were washed with PBS and resuspended inRPMI-1640 media supplemented with benzylpenicillin 100000 U.U, andstreptomycin 100 mg·L⁻¹. The cell viability and concentration weredetermined by trypan blue exclusion.

[0170] Peritoneal exudate cells were induced in BALB/C mice by anintraperitoneal injection of 0.5 ml of 3% TG. After 4 days, theperitoneal exudates cells were harvested by sterile lavage.

[0171] THP-1 (American Type Culture Collection, Manassas, Va.) is ahuman monocytic leukemia. THP-1 cells were maintained in suspensionculture in RPMI1640 medium supplemented with 10% FBS. Cultures weremaintained at 37° C. in a humidified atmosphere of 5% CO₂ in air andwere subculture at {fraction (1/10)} dilution every 5-6 days.

[0172] [³H]-thylnidine Incorporation to the Splenic Lymphocytes

[0173] Mouse splenic lymphocytes were cultured in vitro in RPM11640supplemented with 10% FBS. Cells were incubated in a 96-well plate at1×10⁵cells/200 μl/well in a humidified CO₂ incubator at 37° C. for 48hours with 5 μg/ml of Con A or 10 μg/ml LPS in the presence or absenceof various concentrations of DZ2002. After 40 hour incubation, cellswere pulsed with 0.5 μCi/well of [³H]-thymidine and cultured for another8 hours. The cells were then harvested onto glass fiber filters and theincorporated radioactivity was counted using a Beta Scintillator(MicroBeta Trilux, PerkinElmer Life Sciences).

[0174] MTT Assay of the Splenic Lymphocytes

[0175] Cytotoxicity was assessed with MTT assay. Mouse spleniclymphocytes were incubated in a 96-well plate at 9×10⁴cells/180 μl/wellin a humidified CO₂ incubator at 37° C. for 48 hours in the presence orabsence of various concentrations of DZ2002. Fifteen (15) μl of 5 mg/mlof MTT was pulsed 4h prior to end of the culture (total 190 μl), andthen 80 μl solvent (10% SDS, 50% N,N-dimethy formamide, PH7.2) wasadded. Incubate for 7 h and read OD₅₉₀ at a microplate reader (Bio-radModel 550 Japan).

[0176] Cytokine Production

[0177] Murine splenic mononuclear cells (5×10⁶) were cultured in 24-wellplates in a volume of 2 ml/well in the presence of Sac (1:10000), ConA(5 ug/ml) or LPS (10 ug/ml) in the presence or absence of variousconcentrations of DZ2002. After 24 h, cell-free supernatant wascollected and frozen at −20° C. The concentrations of IL-12p40,IL-12p70, IL-10 and TNF-α were determined in an ELISA specific formurine cytokines.

[0178] Murine peritoneal exudate cells (6.25×10⁵) were cultured in24-well plates in a volume of 1 ml/well for 2 hours. In adherent cellswere washed by ice cold RPMI 1640 and adherent cells were culture in avolume of 2 ml/well in the presence of IFN-γ (2.5 ng/ml) and LPS (1μg/ml) in the presence or absence of various concentrations of DZ2002.After 24 h, cell-free supernatant was collected and frozen at −20° C.The concentrations of IL-12p40, IL-12p70, IL-10 and TNF-α weredetermined by ELISA.

[0179] THP-1 cells (6×10⁵) were cultured in 24-well plates in a volumeof 2 ml/well in the presence of 1.2% and in the presence or absence ofvarious concentrations of DZ2002. After 24 h, IFN-γ (500 U/ml) was addedand another 16 h later, LPS (1 μg/ml) was added. Cell-free supernatantwas collected after 24 h and frozen at −20° C. The concentrations ofIL-12p40, IL-12p70, IL-10 and TNF-α were determined for ELISA.

[0180] Quantitative Hemolysis of Sheep Red Blood Cells (QHS) Assay

[0181] Female Babl/c mice were immunized by intraperitoneal injectionwith 0.2 ml of 16.7% of SRBC on day 4. Vehicle, Dexamethasone and DZ2002were administrated on each group (n=6) by intraperitoneal injection on 7consecutive days of 1-7. On day8, mice were sacrificed and made a mixedsuspension of spleen cells of 2×06 cells/ml. 1 ml of cell suspension wasincubated with 1 ml of 0.5% SRBC and 1 ml of 1:10 dilution of guinea pigcomplement for 1 h at 37° C., then centrifuged (3 min, 3000 g) anddetermined the supernatant hemolysis at 413 nm, according to Simpson etal, J. Immunol. Methods., 21(1-2):159-65.(1978) with some modifications.Each group was triplicated.

[0182] Mixed Lymphocyte Reaction (MLR) Proliferation Assay.

[0183] Balb/c mouse spleen cells were prepared in 10⁷cells/mlsuspension, cultured 2 h with 50 μg/ml of mitomycin. Then cells werewashed and cultured together with fresh C57/B6 mice splenocytes equallyin a final concentration of 1.0×10⁶cells/ml in the presence or absenceof various concentrations of DZ2002. After 48 hour incubation, cellswere pulsed with 0.5 μCi/well of [³H]-thymidine and cultured for another24 hours. The cells were then harvested onto glass fiber filters and theincorporated radioactivity was counted using a Beta Scintillator(MicroBeta Trilux, PerkinElmer Life Sciences).

[0184] DNFB-Induced Delayed Type Hypersensitivity (DTH) Response

[0185] Female Balb/c mice were sensitized with 20 μl of 0.6% DNFBdissolved in acetone-olive oil (4:1) on each hind foot on day0 and 1. Onday 7 mice were challenged with 10 μl of 0.5% DNFB on both sides of leftear, methods according to Phanuphak (1974) with some modifications.Vehicle, CsA, and DZ2002 (1, 3, 10 mg/kg) were administrated on eachgroup (n=10) by intraperitoneal injection on 1 hour before and 12 hours,24 hours after the challenge. Ear swelling was expressed as differencebetween the weight of the left and right ear patches made by a specific8-mm punch 30 h after the challenge.

[0186] Flow Cytometry

[0187] Murine peritoneal exudate cells or THP-1 cells were washed incold PBS (staining buffer, containing 0.1% NaN₃, 1% FBS, PH 7.2). Cellswere resuspended at 2.0×10⁷/ml in cold staining buffer. Optimalconcentrations of each fluorochrome-labeled antibody were added to 50 μLcells. Fc receptors were blocked using 10 μL normal mouse serum. Cellswere incubated in the dark at 4° C. for 30 min, washed twice with 2.0 mLstaining buffer and resupended in 0.5 mL of PBS, PH 7.2. Cells werestored in the dark at 4° C. and analyzed on a FACScan flow cytometer(Becton Dickinson, San Jose, Calif.). Data were analyzed by means ofCellQuest™ Software (Becton Dickinson, San Jose, Calif.).

[0188] Statistical Analysis

[0189] Results were expressed as x±s, independent two-tailed t-test wasperformed and P values less than 0.05 were considered to be significant.Each experiment was repeated at least three times.

[0190] Results

[0191] Inhibition of [³H]-Thymidine Incorporation to the SplenicLymphocytes by AdoHcy Hydrolase Inhibitors

[0192] After 48 h of culture, DZ2002 (0.1-10 μmol·L⁻¹) have no effectson the lymphocytes proliferation induced by ConA. DZ2002 (10 μmol·L⁻¹)inhibited lymphocytes proliferation induced by LPS.

[0193] Effect of DZ2002 on IL-10, IL-12P40 and IL-12P70 Production fromSac Stimulated Murine Splenocytes

[0194] Sac stimulation induced marked increasing of IL-10, IL-12P40 andIFN-γ production from murine splenocytes compared with restingsplenocytes. DZ2002 (μmol·L⁻¹) dose dependently inhibited IL-12P40,IL-12P70 and TNF-α release, but have no effect on IL-10 production fromSac stimulated splenocytes. (data not shown).

[0195] Effects of DZ2002 On Quantitative Hemolysis of Sheep Red BloodCells (QHS) Assay

[0196] Quantitative hemolysis of SRBC is a model of primary antibodyproduction in response to antigenic stimulation. As FIG. 3 shows,consecutively 7-day intraperitoneal injection of DZ2002 inhibited 24.5and 18.4% of QHS at doses of 0.08 and 2 mg/kg respectively, comparedwith 38.1% of that of 5 mg/kg Dethamethasone (p<0.05 for All experimentgroups compared with Vehicle control group) (FIG. 1).

[0197] DZ2002 Suppress T Cell Proliferation In Mixed Lymphocyte Reaction

[0198] Mitomycin-treated Balb/c (H-2^(d)) spleen cell were applied asallogeneic stimulator to C57BL/6(H-2^(b)) spleen cells proliferation.DZ2002 had a strong suppression to MLR with 40.2, 36.9 and 42.3% atdoses of 0.1, 1 and 10 μmol/L respectively for 3-day culture. (FIG. 2).

[0199] DZ2002 Have No Cytotoxicity In Spleen Cell

[0200] In two days of culture, 0.1-10 mmol/L DZ2002 showed nocytotoxicity to spleen cells. The OD values of cells incubated withDZ2002 have no difference with that of the control. (FIG. 3).

[0201] DZ2002 Reversed the Suppression of Mouse Dth Response Induced byEthanol Consumption

[0202] Nine mice were prepared for each group. Mice were sensitized with0.5% DNFB solution (20 ul) in absolute acetone/olive oil (4:1) on eachhind foot on day 0 and 1. Five days after initial sensitization, micewere challenged with 0.2% DNFB (10 ul) on both sides of left ear underlight Metofane anesthesia. The right ear was treated with vehicle alone.DZ2002 were orally administered to the mice 1 h before DNFB challenge.The degree of ear swelling was measured 24 h after challenge using a earpuncher and an analytic balance to measure the weight (mg). Results wereexpressed as the difference between the weight of the left and the rightear. Spleens were taken from four mice in each group after themeasurement of the ear swelling and frozen until analysis.

[0203] Effects of DZ2002 On the Expression of MHC-II On Resident and TGInduced Peritoneal Cells

[0204] MHC-II expression by peritoneal macrophages was assessedfollowing a 48-h incubation with media alone or with IFN-γ at 100 U/ml.Cells incubated with IFN-γ in the present of 0.1 and 1 μmol/L DZ2002enhance the levels of MHC-II expression of resident peritoneal cells and10 μmol/L DZ2002 reduce the levels of MHC-II expression.(data not shown)As reflected in the TG induced peritoneal cells, 1 and 10 μmol/L DZ2002decrease the Mac-1⁺ percentage when incubate with media alone. And thelevel of MHC-II expression of cells incubated with IFN-γ wasdose-dependently decreased in the presence of 0.1-10 μmol/L DZ2002.(data not shown).

[0205] Effect of DZ2002 on IL-10, IL-12P40 and TNF-α Production from TGInduced Peritoneal Cells

[0206] Cytokines produced by peritoneal macrophages were assessedfollowing a 24-h incubation with IFN-γ at 25 U/ml and LPS at 1 μg/ml.Resident peritoneal cells produce low levers of cytokines except forsome IL-10 with incubated with IFN-γ and LPS (data not shown). As for TGinduced peritoneal macrophages, DZ2002 inhibited IL-12P40 and TNF-αrelease, but have no effect on IL-10 production in the dose of 0.1-10μmol/L (FIG. 5).

[0207] DZ2002 Inhibits Expression of MHC-II, CD80 and CD86 on THP-1Cells

[0208] MHC-II, CD80 and CD86 expression by THP-1 cells was assessedfollowing a 48-h incubation with media alone or with IFN-γ at 100 U/ml.Cells incubated with IFN-γ in the present of 10 μmol/L DZ2002 modestlyreduce the levels of MHC-II expression of THP-1 cells and 0.1-10 μmol/LDZ2002 reduce the levels of CD80 and CD86 expression by andose-dependently way. (FIG. 6A-C).

[0209] Effect of DZ2002 on IL-10, IL-12P40 and TNF-α Production FromTHP-1 Cells

[0210] Cytokines produced by THP—I cells were assessed following a 24-hincubation with IFN-γ at 500 U/ml and LPS at 1 μg/ml. As FIG. 7 shows,DZ2002 inhibited IL-12P40 and TNF-α release, in the dose of 0.1-10μmol/L.

[0211] The above examples are included for illustrative purposes onlyand are not intended to limit the scope of the invention. Manyvariations to those described above are possible. Since modificationsand variations to the examples described above will be apparent to thoseof skill in this art, it is intended that this invention be limited onlyby the scope of the appended claims.

What is claimed is:
 1. A compound or a pharmaceutically acceptable saltthereof, having the formula (I):

wherein Z is selected from the group consisting of carbon and nitrogen,R1 and R2 are the same or different, and are selected from the groupconsisting of hydrogen, hydroxy, alkyl, cycloalkyl, alkenyl, alkoxy,amino, aryl, heteroaryl, and halogen; R3 and R4 are the same ordifferent and are selected from the group consisting of hydrogen, alkyl,acetyl, alkenyl, aryl, and heteroaryl; X is selected from the groupconsisting of oxygen, nitrogen, and sulfur; and Y is selected from thegroup consisting of hydrogen, a C₁₋₁₀ alkyl group, alkenyl, vinyl, aryl,and heteroaryl, provided that the compound is not(4-adenine-9-yl)-2-hydroxybutanoic acid.
 2. The compound of claim 1,wherein R1, R2, R3, and R4 are hydrogen.
 3. The compound of claim 1,wherein X is oxygen.
 4. The compound of claim 1, wherein Y is a C₁₋₁₀alkyl group.
 5. The compound of claim 1, wherein R1, R2, R3, and R4 arehydrogen, X is oxygen, and Y is a C₁₋₁₀ alkyl group.
 6. The compound ofclaim 1, wherein the β carbon of said compound has a configurationselected from the group consisting of S, R, and a racemic mixturethereof.
 7. The compound of claim 1, wherein said compound has a K_(i)value less than 100 nM for a mammalian S— adenosyl-L-homocysteine (SAH)hydrolase in a biological medium.
 8. The compound of claim 7, whereinsaid mammal is human.
 9. The compound of claim 1, wherein said compoundhas a K_(i) value between about 1 nM and about 100 nm for a mammalianSAH hydrolase in a biological medium.
 10. The compound of claim 9,wherein said mammal is human.
 11. A pharmaceutical compositioncomprising a compound of claim 1 and a pharmaceutically acceptableexcipient.
 12. The pharmaceutical composition of claim 11, wherein saidcompound has a K_(i) value less than 100 nM for a mammalian SAHhydrolase in a biological medium.
 13. The pharmaceutical composition ofclaim 12, wherein said mammal is human.
 14. The pharmaceuticalcomposition of claim 11, wherein said compound has a K_(i) value betweenabout 1 nM and about 100 nM for a mammalian SAH hydrolase in abiological medium.
 15. The pharmaceutical composition of claim 14,wherein said mammal is human.
 16. The pharmaceutical composition ofclaim 11, wherein said composition is formulated for oral, parenteral,intranasal, topical, or injectable administration.
 17. Thepharmaceutical composition of claim 11, wherein said composition isformulated in a solid or liquid dosage form.
 18. The pharmaceuticalcomposition of claim 11, wherein said composition is formulated for oraladministration in a dosage ranging from about 0.1 to about 20 mg/kg perday.
 19. The pharmaceutical composition of claim 11, wherein saidcomposition is formulated for injectable administration in a dosageranging from about 0.1 to about 20 mg/kg per day.
 20. The pharmaceuticalcomposition of claim 19, wherein said injectable administration isselected from the group consisting of intracavemous injection,subcutaneous injection, intravenous injection, intramuscular injection,and intradermal injection.
 21. A kit, comprising an effective amount ofsaid composition of claim 11, and an instruction means for administeringsaid composition.
 22. A method for reversibly inhibiting activity of aS-adenosyl-L-homocysteine (SAH) hydrolase in a mammal, comprisingadministering to a mammal to which such reversible inhibition is neededor desirable, an effective amount of a compound or a pharmaceuticallyacceptable salt thereof, having the formula (I):

wherein Z is selected from the group consisting of carbon and nitrogen,R1 and R2 are the same or different, and are selected from the groupconsisting of hydrogen, hydroxy, alkyl, cycloalkyl, alkenyl, alkoxy,amino, aryl, heteroaryl, and halogen; R3 and R4 are the same ordifferent and are selected from the group consisting of hydrogen, alkyl,acetyl, alkenyl, aryl, and heteroaryl; X is selected from the groupconsisting of oxygen, nitrogen, and sulfur; and Y is selected from thegroup consisting of hydrogen, a C₁₋₁₀ alkyl group, alkenyl, vinyl, aryl,and heteroaryl, thereby reversibly inhibiting the activity of SAHhydrolase in said mammal.
 23. The method of claim 22, wherein saidmammal is human.
 24. The method of claim 23, wherein said mammal issuspected of having a disease selected from the group consisting ofhemorrhagic viral infection, autoimmune disease, autograft rejection,neoplasm, hyperhomocysteineuria, cardiovascular disease, stroke,Alzheimer disease, and diabetes.
 25. The method of claim 24, whereinsaid hemorrhagic viral infection is caused by a virus selected from thegroup consisting of a Bunyaviridaea, a Filoviridae, a Flaviviridae, andan Arenaviridae virus.
 26. The method of claim 25, wherein saidFiloviridae virus is Ebola virus.
 27. The method of claim 22, whereinsaid administering step comprises administering an effective amount ofsaid compound in the treatment of an autoimmune disease in said mammal.28. The method of claim 22, wherein said administering step comprisesadministering an effective amount of said compound in the treatment ofallograft rejection in said mammal.
 29. The method of claim 22, whereinsaid administering step comprises administering an effective amount ofsaid compound for lowering homocysteine in said mammal.
 30. The methodof claim 22, wherein said administering step comprises administering aneffective amount of said compound in the treatment of a neoplasm in saidmammal.
 31. The method of claim 30, wherein said neoplasm is selectedfrom the group consisting of adrenal gland, anus, auditory nerve, bileducts, bladder, bone, brain, breast, bruccal, central nervous system,cervix, colon, ear, endometrium, esophagus, eye, eyelids, fallopiantube, gastrointestinal tract, head and neck, heart, kidney, larynx,liver, lung, mandible, mandibular condyle, maxilla, mouth, nasopharynx,nose, oral cavity, ovary, pancreas, parotid gland, penis, pinna,pituitary, prostate gland, rectum, retina, salivary glands, skin, smallintestine, spinal cord, stomach, testes, thyroid, tonsil, urethra,uterus, vagina, vestibulocochlear nerve, and vulva neoplasm.
 32. Themethod of claim 31, wherein said neoplasm is selected from the groupconsisting of breast, ovary, stomach, prostate, colon and lung cancer.33. The method of claim 22, wherein said compound or a pharmaceuticallyacceptable salt thereof is not (4-adenine-9-yl)-2-hydroxybutanoic acid.34. A combination, comprising: a) an effective amount of a compound or apharmaceutically acceptable salt thereof, having the formula (I):

wherein Z is selected from the group consisting of carbon and nitrogen,R1 and R2 are the same or different, and are selected from the groupconsisting of hydrogen, hydroxy, alkyl, cycloalkyl, alkenyl, alkoxy,amino, aryl, heteroaryl, and halogen; R3 and R4 are the same ordifferent and are selected from the group consisting of hydrogen, alkyl,acetyl, alkenyl, aryl, and heteroaryl; X is selected from the groupconsisting of oxygen, nitrogen, and sulfur; and Y is selected from thegroup consisting of hydrogen, a C₁₋₁₀ alkyl group, alkenyl, vinyl, aryl,and heteroaryl; and b) an effective amount of a compound selected fromthe group consisting of an anti-hemorrhagic viral infection agent, animmunosuppressant, a homocysteine lowering agent, and an anti-neoplasmagent.
 35. The combination of claim 34, wherein said anti-hemorrhagicviral infection agent inhibits interleukin-1 (IL-1), tumor necrosisfactor (TNF), or a combination thereof.
 36. The combination of claim 34,wherein said anti-hemorrhagic viral infection agent is selected from thegroup consisting of an anti-viral vaccine, an anti-viral antibody, aviral-activated immune cell, and a viral-activated immune serum.
 37. Thecombination of claim 34, wherein said immunosuppressant is selected fromthe group consisting of cyclosporine, tacrolimus, an adrenocorticalsteroid, azathioprine, mycophenolate, cyclophosphamide, methotrexate,chlorambucil, vincristine, vinblastine, dactinomycin, an antithymocyteglobulin, muromonab-CD3 monoclonal antibody, Rh₀(D) immune globulin,methoxsalen, and thalidomide.
 38. The combination of claim 34, whereinsaid homocysteine lowering agent is selected from the group consistingof vitamin B₆, vitamin B₁₂ and folate.
 39. The combination of claim 34,wherein said anti-neoplasm agent is selected from the group consistingof an anti-angiogenic agent, an alkylating agent, an antimetabolite, anatural product, a platinum coordination complex, an anthracenedione, asubstituted urea, a methylhydrazine derivative, an adrenocorticalsuppressant, a hormone, an antagonist, an oncogene inhibitor, a tumorsuppressor gene or protein, an anti-oncogene antibody, and ananti-oncogene antisense oligonucleotide.
 40. The combination of claim34, further comprising a pharmaceutically acceptable carrier orexcipient.
 41. The combination of claim 34, wherein said compound or apharmaceutically acceptable salt thereof is not(4-adenine-9-yl)-2-hydroxybutanoic acid.
 42. A method for reversiblyinhibiting activity of a SAH hydrolase in a mammal, comprisingadministering to a mammal to which such reversible inhibition is neededor desirable, an effective amount of a combination, wherein thecombination comprises: a) an effective amount of a compound or apharmaceutically acceptable salt thereof, having the formula (I):

wherein Z is selected from the group consisting of carbon and nitrogen,R1 and R2 are the same or different, and are selected from the groupconsisting of hydrogen, hydroxy, alkyl, cycloalkyl, alkenyl, alkoxy,amino, aryl, heteroaryl, and halogen; R3 and R4 are the same ordifferent and are selected from the group consisting of hydrogen, alkyl,acetyl, alkenyl, aryl, and heteroaryl; X is selected from the groupconsisting of oxygen, nitrogen, and sulfur; and Y is selected from thegroup consisting of hydrogen, a C₁₋₁₀ alkyl group, alkenyl, vinyl, aryl,and heteroaryl; and b) an effective amount of a compound selected fromthe group consisting of an anti-hemorrhagic viral infection agent, animmunosuppressant, a homocysteine lowering agent, and an anti-neoplasmagent, thereby reversibly inhibiting said activity of SAH hydrolase insaid mammal.
 43. The method of claim 42, wherein said compound or apharmaceutically acceptable salt thereof is not(4-adenine-9-yl)-2-hydroxybutanoic acid.
 44. A kit, comprising aneffective amount of a combination of claim 34 and an instruction meansfor administering said combination.
 45. A method for identifying acandidate inhibitor compound capable of inhibitingS-adenosyl-L-homocysteine hydrolase (SAH) activity, comprising the stepsof: a) constructing a computer model of the SAH binding pocket; b)screening a plurality of compounds having the structure

wherein Z is selected from the group consisting of carbon and nitrogen;and c) identifying a compound that computationally binds to said bindingpocket.
 46. The method of claim 45, further comprising the step ofassaying said compound to determine the ability of said compound toinhibit SAH activity.
 47. A method for reversibly inhibiting activity ofa SAH hydrolase, comprising contacting a SAH hydrolase with an effectiveamount of said compound of claim 1 to reversibly inhibit the activity ofsaid SAH hydrolase.
 48. A method for reversibly inhibiting activity of aSAH hydrolase, comprising contacting a SAH hydrolase with an effectiveamount of said combination of claim 34 to reversibly inhibit theactivity of said SAH hydrolase.
 49. The method of claim 22, which isused to inhibit lymphocyte proliferation, to inhibit production and/orrelease of IL-12P40, IL-12P70 and TNF-α or to inhibit primary antibodyproduction in the mammal.
 50. The method of claim 23, wherein saidmammal is suspected of having a disease selected from the groupconsisting of inflammatory Bowel disease, multiple sclerosis andautoimmune neuritis.